Vertebrate Biotechnology Flashcards
What is pronuclear injection and what are some advantages?
The simplest transgenic method
Was discovered in the 1980s
Widely applicable across species for adding genes in random locations
Lead to the development of other methods
Is the standard method for adding genes
Cheap, effective, proven, useful and can be used cross species
Describe how pronuclear injection works
A small suction pipette holds a newly fertilised mouse egg in place using negative pressure
A glass pipette injects picolitres of DNA containing the DNA fragments of the transgene penetrates the egg cell membrane then the haploid nuclei from the egg and sperm which haven’t combined into a diploid yet
They are then grown in culture to a morula
The morula is then implanted into pseudo-pregnant females (have been mated with sterile males and have the needed hormonal changes)
The pups are then tested for presence of the transgene and then are bred with heterozygotes to produce a homozygous mouse
They are then bred to multiple generations to ensure successful uptake of the transgene in the germ cells and for any adverse side effects
Why are mouse eggs used?
The standard model for transgenics
Breed rapidly
Have short generations
Are small
Give birth to 6-8 pups
What DNA is injected in pronuclear injection?
A promoter which has been chosen according to the regulations of the application e.g. constitutive promoters have strong, constant expression whereas tissue specific promoters are regulated and their activity can be precisely controlled
An intron which contains the binding sites for transcription factors
An open reading frame containing the cDNA or DNA sequence
A polyadenylation site which allows the polyA tail to be attached to mRNA and is needed for the export of mRNA from the nucleus and translation
What form should the transgene be in before injection?
Linear as they integrate into the genome at a higher frequency than circular plasmids so circular plasmids are cut using a restriction enzyme
Multiple linear copies are injected into the egg
What are some problems with pronuclear injection?
Can only be used to add genes not modify or take away
Ectopic insertions as there is no control over where in the chromosome it is inserted
Multiple copies can join together in chains
Position effects of flanking chromosomal sequences can lead to silencing or enhancement effects thus the expression of the transgene will vary depending upon its position in the genome
The transgene is not always consistently inherited and could disrupt other genes if inserted incorrectly i.e. in a regulatory sequence for another gene
What is homologous recombination?
Developed in the late 1980’s
Enables the precise modification of specific genes in embryonic stem cells
A single copy of the modified gene is modified in its normal chromosomal environment
Relies on the natural process of homologous recombination (exchange of DNA segment between DNA strands in chromosomes)
Applicable only to mice
What are embryonic stem cells and what are the advantages of using them?
Also called a blastocyst and contains ~150 cells and are pluripotent
Are formed shortly after fertilisation thus are developmentally flexible
Embryonic stem cells from the blastocyst inner mass are cut out and grown in culture
Are capable of dividing into any cell type
Have a higher rate of homologous recombination than other cells
Describe HR knockins
HR allows a transgene to be inserted into a precise position in a chromosome
Describe HR knockouts
Insert and expression of a marker gene in a position that results in a natural gene being inactivated
An example is the introduction of neoR marker gene which makes cells survive exposure to neomycin thus positive selection (this is rare as it is more likely to randomly insert)
A second marker such as thymidine kinase is incorporated which kills the cells thus negative selection
Describe how a knockout mouse is made
A mouse blastocyst has the embryonic stem cells removed from its inner mass and the targeting vector is introduced by electroporation (which opens the membrane channels) or lipofection (transgene forms complex with lipid and gently enters cells)
Positive/negative selection screens for embryonic stem cells with the targeting vector
The targeted embryonic stem cells are injected into blastocysts and mixed with natural embryonic stem cells (the blastocyst comes from a mouse with different fur colour than the original blastocyst) and is then injected into foster mothers
This results in the birth of chimeric mice with the transgene (have the cells of 2 genomes)
A normal mouse is mated with a chimeric mouse to produce desired heterozygous progeny
Further cross breeding is required for homozygous knockouts as only one allele on one chromosome has been modified
Why is it not possible to grow a whole mouse from embryonic stem cells?
The embryonic stem cells are pluripotent not totipotent and as such are too developed
Need to return modified stem cells to a blastocyst and hope some of them differentiate into germ cells to the modification is inherited in future generations
Why are knockouts important?
Allows for the studying of gene function and the mechanisms of diseases
Describe how Dolly the sheep was made
Donor cells were removed from the mammary gland (fully differentiated cells) of a Finn Dorset Ewe (white face) and starved in a low-nutrient culture medium to arrest the growth cycle
An unfertilised egg cell is donated by a Scottish Blackface Ewe and has the nucleus removed to produce and enucleated egg cell
The donor cells and the enucleated egg cell are fused with electrical pulses and will behave as if it is a newly fertilised egg
The fused cell undergoes cell division and forms and embryo which is then implanted into a Scottish Blackface Ewe surrogate mother
Describe how Dolly the sheep was made
Nuclear transfer
Took 177 attemps
Donor cells were removed from the mammary gland (fully differentiated cells) of a Finn Dorset Ewe (white face) and starved in a low-nutrient culture medium to arrest the growth cycle
An unfertilised egg cell is donated by a Scottish Blackface Ewe and has the nucleus removed to produce and enucleated egg cell
The donor cells and the enucleated egg cell are fused with electrical pulses and will behave as if it is a newly fertilised egg
The fused cell undergoes cell division and forms and embryo which is then implanted into a Scottish Blackface Ewe surrogate mother
Dolly was born with a white face thus taking on the phenotype of the donor cell not the egg donor or surrogate mother phenotype
Why is the rat an important model animal?
Was the first mammal bred for research
Is the most widely used mammalian biomedical model and is the standard model for drug testing in clinical trials of human drugs and physiology whereas mice are used for genetics
Are larger than mice which makes the measurement of organs simpler
However rat embryonic stem cells are hard to culture so HR cannot happen
What are ZFNs?
Zinc finger nucleases are hybrid fusion proteins which are used to cut DNA
FokI nuclease cleavage domain is combined with several zinc finger domains which have been engineered to bind to a specific target sequence
How do ZFNs work?
FokI cleavage domain requires dimerisation to break the double strand and 2 ZFNs are designed to target sequences on each strand separated by a 6 bp cleavage site
The double strand break activates the DNA repair response and in 70% of cases, non homologous end joining (NHEJ) will occur involving short insertions or deletions to disrupt genes (knockout) and in the other 30% of cases homologous recombination (HR) will occur if a template sequence is introduced (knockin)
Describe the research conducted in the first ZFN transgenic rat paper
Knockout rats were produced via embryo microinjection of ZFNs that targeted immunoglobin heavy chain μ locus (IgM) which is needed for B lymphocyte survival
Used a promoter that was only activated briefly in the early embryo (CAG promoter)
The vector was not incorporated into the genome but instead injected into the egg as a circular plasmid
Describe how TALENs was used in cattle gene editing tests
Knockout of the myostatin gene (inhibits muscle development) in cattle and sheep using TALENs (engineered restriction enzymes with DNA binding and cleavage domains similar to ZFNs) injected into the egg cytoplasm which caused muscular hypertrophy in knockouts
What are some unintended effects of CRISPR/Cas9?
Off target effects where there are unintended DNA breaks in regions with a similar sequence to the target site (this can be controlled using bioinformatic software of using modified Cas enzymes)
Mosaicism and multiple alleles where the editing persists beyond the one-cell stage and multiple alleles are created (extensive breeding and genotyping is needed to isolate the animals with the desired alleles)
Integration of the template vector when creating knockins
Why were transgenic fish produced?
Increasing world demand for fish
Little selective breeding had been done in fish
Fish populations are decreasing
The potential for aquaculture (fish populations bred in pens off the coast for commercial production)
How were the first transgenic fish made?
In 1985, goldfish expressing human growth hormone were made via direct injection of DNA into fertilised egg cytoplasm as the pronucleus was too hard to see
How were AquAdvantgage salmon made?
Ocean pout antifreeze protein gene (AFP) 5’ promoter and 3’ terminator was linked to salmon growth hormone
The promoter allowed the growth hormone to be produced outside the pituitary gland and during all seasons
The growth rate not the final size was affected so transgenic salmon reached market size 1 year and required less food
Was under development for 30 years and had a long wait for approval from FDA due to non-existent regulations and pressure groups
What were some of the sustainability claims made by AquaBounty?
The salmon ate 25% less than conventional farmed fish and had a high feed conversion compared to traditional farm animals
Inland production facility in Indiana prevented escape and mating with wild-type salmon which would cause declines in the natural population
Salmon were made sterile to prevent affecting natural populations in case of escape
The inland facility also allowed recycling and treatment of the water to prevent diseases, parasites and pollution
The fish were produced closer to the consumer than wild or farmed fish so there were reduced carbon dioxide emissions
Describe how xenotransplantation works
Lack of supply from human donors means that animal organs can be made suitable for implantation into human hosts
Pigs are used as they are efficient breeders, have similar organ size to humans and have compatible life expectancies
Genes involved in rejection can be manipulated as the human immune system would recognise the organ as foreign
Describe how gene knockout in pigs by nuclear transfer can aid xenotransplantation
Alpha 1,3 galactosyl transferase is not present in humans but adds a carbohydrate antigen to pig cells thus would be attacked by human immune system
Knockout in pigs by HR and nuclear transfer using cultured foetal fibroblasts were used
Describe the use of human embryonic stem cells in therapeutic cloning
Human embryonic stem cells were isolated in 1988
Nuclear transfer of somatic cell into a enucleated donor oocyte then grow ESC in culture
Could grown patient’s own cells (to replace diseased cells/tissues) or correct genetic defects
Are immunologically compatible for transplant
What are iPS cells?
Induced pluripotent stem cells
Reverse the differentiation by administering 4 transcription factors in viral vectors
Avoids the need to clone and kill human embryos to collect ESC
How are iPS made?
Skin cells are collected
Using Oct4, Sox2, Klf4 and c-Myc viruses to reprogram the skin cells into ESC like iPS cells
This produces genetically identical iPS cells that will need to have mutations corrected and differentiated
They can then be transplanted into sick individuals
E.g. differentiate into blood stem cells and inject into patient with sickle cell anaemia
